EP0514421B1 - Process for depleting viruses in solutions and for determining the depletion rate of the viruses - Google Patents

Abstract

To deplete viruses in organic material, the material to be purified is conveyed through an ultrafilter or an ultrafiltration unit the depletion rate of which is previously determined. The filter or filtration unit is charged with viruses of the family Leviviridae and the viral count is determined before and after filtration and used to derive the depletion rate. The virus depletion can be monitored during the process by following the depletion of a marker.

Description

The invention relates to a method for depleting viruses and for determining the depletion rate of viruses in organic material.

Medicinal products derived from animal or human cell cultures, organs or blood are potentially contaminated with animal or human pathogenic viruses. With the wide range of viruses with which the sample can be contaminated, it is impossible to test the starting material for all viruses that may be present. For some virus groups there is also no reliable or sufficiently sensitive detection method. A cleaning or inactivation process must therefore be carried out, by means of which pathogenic viruses are depleted, so that no problems are to be expected even in the case of a massive infection of starting material or intermediate products. The cleaning process or inactivation process should be so efficient that viruses can be depleted by rates of up to 10 ¹² .

In order to check these depletion rates, samples of the material have to be spiked with viruses in extreme concentration ranges and the decrease in the virus titer has to be checked. Since viruses can differ significantly from one another in their physicochemical behavior, the material for the manufacturing process must be exposed to a spectrum of at least four different virus types. This requires considerable effort, since potentially human-pathogenic viruses must also be used. These complex processes are, for example, in Heimburger, Schwinn, Gratz, Lüben, Kumpe and Herchenhahn, factor VIII concentrate, highly purified and heated in solution, Arzneimittelforschung 31 (1981), 619-622; Mauler and Hilfenhaus; Inactivation of viruses in factor VIII concentrate by heating in solution; Drug Research 34 (1984), 1524-1527; Hilfenhaus, Mauler, Friis and Bauer; Safety of human blood products; inactivation of retroviruses by heat treatment at 60 _° C, Proc. Soc. Exp. Biol. Med., 178 (1985), 580-584, for obtaining coagulation factors from human serum.

Sterile filtration has long been used in the manufacture of pharmaceuticals to remove bacteria and is considered a safe decontamination process for these potential pathogens. The manufacturer applies random samples to the sterile filter with the smallest bacterium Pseudomonas diminuta known outside the groups of mycoplasma and L-shape bacteria. If a certain depletion rate for sterile filtration can be detected for this bacterium in the "bacteria challenge test", the production batch is considered safe. Such a method is described in Wallhäußer, Praxis der Sterilisierung, Thieme Verlag, Stuttgart, 1988, pages 324 ff.

Filtration methods can also be used to remove viruses, the filters used having to retain molecules and particles of more than 1 million Daltons. Such ultrafilters are available in various designs, but - in contrast to sterile filters - they are not absolute filters; d. H. Molecules and particles over 1 million Daltons are not retained absolutely, but only to a large extent. The retention rate is not only dependent on the filter type, it can even differ from production batch to production batch. Ultrafilters have therefore not yet been used for virus removal, but have at most contributed to total virus depletion in a production process consisting of several steps (Werner and Langlius-Gane, Meeting the Regulatory Requirements for Pharmaceutical Production of Recombinant DNA Derived Products, Arzneimittel-Forschung, Vol. 39 ( 1989), 108-111). This unreliability of ultrafilters is due to the filter production process. With ultrafilters, which are provided for a defined molecular weight exclusion, ever larger micropores can occur, which, for. B. can be permeable to viruses. In addition, ultrafilters, like microfilters, cannot be tested for leaks using the so-called bubble point method.

It was therefore an object of the invention to provide a method for the depletion of viruses, with which a depletion by at least 12 powers of ten is achieved. In addition, it was an object of the invention to provide a method with which the depletion rate for viruses can be determined simply and precisely and which provides information about which filtration method and by how many filtration steps a depletion deemed safe is achieved.

This object is achieved by a method for the depletion of viruses in solutions, which is characterized in that the solution to be cleaned is passed through a filter or a filtration unit, the depletion rate of which has previously been determined by the filter or the filtration unit with viruses of the Leviviridae family was applied and the titer of the viruses was determined before and after filtration and the depletion rate was determined therefrom. According to the invention, it is also possible to use other comparatively small bacteriophages. These bacteriophages are preferably detectable through simple holes in a bacterial lawn.

According to the invention, it is possible to carry out ultrafiltration as the only method for secure virus depletion without additional cleaning or inactivation steps by controlling the depletion rate of the viruses in the running process. If this depletion rate is determined before and after the filtration of the production batch and is higher than 10 ¹² , virus contamination of the product can be excluded with the greatest possible certainty. So far, validation experiments have only been carried out with animal or even human pathogenic viruses. For safety reasons, the validated filters could therefore no longer be used afterwards. For this reason, new filters with possibly different depletion rates had to be used. Such a validation was therefore always only valid for a single filter and, because of the effort involved, could only be carried out once before or after depletion. In contrast to this, it is possible according to the invention to monitor the virus depletion in the running process, that is to say to carry out an in-process control.

According to the invention, the solution to be cleaned is passed through a filter or a filtration unit, the depletion rate of which was previously determined. By using viruses from the Leviviridae group as test viruses, the depletion rate can be detected safely and without great effort.

• - ein einfaches, innerhalb kurzer Zeit durchführbares und exaktes Validierungsverfahren
• - eine Wiederverwendbarkeit der getesteten Filter ohne zusätzliche Verunreinigung des Produkts. Außerdem können die Leviviridae in extrem hohen Titern von bis zu 10¹⁴ pfu pro ml angezogen werden und durch ein einfaches Plattierungsverfahren selbst in Konzentrationen von 1 pfu pro ml zuverlässig nachgewiesen werden.

With a diameter of 23 nm and a molecular weight of 1.4 million daltons, the Leviviridae are smaller than viruses that are pathogenic to humans or humans (H. Fraenkel-Conrat, The Viruses, Catalog, Characterization and Classification, Plenum Press, 1982). They only attack certain F ⁺ strains of the harmless intestinal bacterium Escherichia coli and, as RNA viruses, are hydrolyzed in 10 mM NaOH within a short time and thereby broken down into their individual molecular components. The smallest animal and / or human pathogenic viruses come from the Picornavirus group, are 27 nm in diameter and 2.5 million Daltons heavy. The Leviviridae are therefore suitable for the validation of size-selecting ultrafilters. The filters can then be washed with 0.1 M NaOH, which also removes pyrogens derived from Escherichia coli. The filters are then ready for production again. The conditions defined for an in-process control are thus fulfilled, namely by:

• - A simple, precise and quick validation procedure
• - Reusability of the tested filters without additional contamination of the product. In addition, the Leviviridae can be grown in extremely high titers of up to 10 ¹⁴ pfu per ml and can be reliably detected by a simple plating method even in concentrations of 1 pfu per ml.

For the determination, the filter is loaded with a virus solution with a titer of over 10 ¹⁰ pfu / ml and the concentration of the phages in the filtrate and in the retained solution is determined. The determination is carried out in a manner known per se (for example using the top agar method from NH Adams (1959), Bacteriophages, Interscience Publishers, New York). For this purpose, for example, the phages are mixed with suitable host bacteria (e.g. BE coli 3300, ATCC No. 19853) and in a layer of 0.6% agar agar on plates with nutrient agar (e.g. 1% bactotrypton, 0.5% Yeast extract, 0.5% NaCl, 0.1 mM CaCl ₂ , 1.5% agar agar) applied. 10 ⁷ to 10 ⁸ bacteria and less than 100 phages should be applied per plate. The plates are then incubated at 37 _° C, whereupon a bacterial lawn develops after 10 hours. Holes in this lawn indicate virus infection and the number of holes gives the virus titer in pfu (plaque-forming units). Since a virus can cause plaque, 1 virus per ml can also be detected with standard agar plates. A virus concentration range of over 10 ¹⁴ to 1 pfu per ml can thus be covered.

The virus depletion rate is then determined by determining the virus concentration in the filtrate and in the solution before the filtration. By determining the virus titer in the concentrate (i.e. the solution retained in front of the filter), it can be calculated whether viruses have been lost due to absorption to the filter or inactivation, which gives the validation process an additional security.

Since the depletion behavior of filtration membranes can differ significantly not only from manufacturer to manufacturer, but also from production batch to production batch, it is essential to determine the depletion rate for viruses for each individual filter.

The filter or filter system provided for cleaning the organic material is preferably checked under precisely defined pressure conditions, which are then also complied with later in the cleaning process.

After the depletion rate has been determined, the filters can then be removed from the bacteriophages and other residues such as pyrogens by simply rinsing with sodium hydroxide solution, and the filters can then be used for the cleaning process of the organic material. This is a further advantage of the method according to the invention, since this would not be possible if animal or human pathogenic viruses were used to determine the depletion rate because of the great risk of contamination with such viruses.

The viruses MS2, f2, f4, Qß, Vk, ST or R17 or comparable strains from the group of Leviviridae are preferably used for the process according to the invention, which are available from H. Fraenkel, Conrat, The Viruses Catalog, Characterization and Classification, Plenum Press , New York, 1982. The bacteriophage fr is particularly preferably used as the test virus, which is deposited with ATCC under the number 15767-B1 and is described in Knolle and Hoffmann-Berling, Virology, Volume 123, 271-273 (1964). This phage consists of a round protein-RNA complex in the form of a polyhedron with a diameter of 23 nm and has a molecular weight of 1.4 million daltons.

In a preferred embodiment, the organic material is purified by ultrafiltration in spiral cartridges. The cartridges are fed by a pump. Before the filtration cartridges are used, the virus depletion factor is determined using the test virus. The test solution, which contains the test viruses in a known amount, is filtered through the cartridge in a tangential flow under precisely defined pressure conditions. The virus titer is then determined in the filtrate according to known methods. The organic material is then cleaned in the same cartridge under the same conditions.

Before being used for the method according to the invention, the test viruses can be raised to a titer of 10 ¹⁴ in a manner known per se. As a host bacterium for the bacteriophages z. B. Escherichia coli 3300, ATCC No. 19853. Culture media for growing phages are known. Suitable is, for example, the medium described by Luria and Bertani, which contains 1 g of distilled water, 10 g of bactotryptone, 5 g of yeast extract and 5 g of NaCl and has a pH of 7.5, which is optionally adjusted with NaOH. To obtain the viruses, they are precipitated from the culture medium by adding a precipitant, for example polyethylene glycol PEG6000. The viruses are then resuspended in buffer and adjusted to the desired titer in the buffer. A Tris-HCl buffer with pH 7.5, for example, which contains 100 mM NaCl under 3 mM CaCl _{2 is} suitable as a buffer. The titer can be determined using the top agar method. This phage suspension is then subjected to the filtration process desired for the organic material. After completion of this filtration, the virus titer is determined and the depletion factor can be calculated from the decrease in the titer. The virus titer is given in the unit "pfu" (plaque-forming-units) and means the number of holes in the bacterial turf caused by the virus infection. The filtration is then repeated after rinsing the cartridge with sodium hydroxide solution and neutralizing by rinsing with distilled water until the desired depletion rate is reached. Likewise, several filters can also be connected in series, through which the filtration is carried out in series. After the depletion factor has been determined, the pyrogens introduced by the phages are removed by rinsing the cartridge with sodium hydroxide solution and are then ready for use in the production process after neutralization.

It has surprisingly been found that the method according to the invention is particularly suitable for using the virus depletion during the production process in the production of aseptic extracts from biological material as a so-called in-process control. It has been found that the depletion of marker substances in the sample to be cleaned correlates with the depletion rate of the viruses. In this way it is possible to follow the virus depletion by determining the depletion of the marker substance.

The procedure according to the invention is such that the depletion rate of the virus and marker is determined, the ratio between the two depletion rates is formed, i.e. that a calibration curve is created therefrom and the virus decrease is monitored in the ongoing process by determining the marker depletion on the basis of the calibration curve.

Easily determinable substances are usually used as marker substances, which are preferably already present in the system to be cleaned. However, it is also possible to add such marker substances to the system to be depleted. Preferred marker substances are proteins, peptides and / or nucleic acids. However, it is also possible to use synthetic substances, in particular oligomers and polymers, as marker substances. Such polymers are known to the person skilled in the art and can be found by simple, simple experiments for the respective system. According to the invention, BSA is used as a particularly preferred marker.

Preferred preparations to be cleaned are usually biological materials, in particular those from plant and animal organisms. Such preparations are preferably obtained from organs, tissues and / or cells. Preferred organs are spleen, thymus and / or bone marrow. However, the method according to the invention is also suitable for cleaning biological material which has been obtained from body fluids or also from bacterial or viral material, in particular from pathogenic material.

In a preferred embodiment according to the invention, the depletion factor is determined using four different viruses.

• Fig. 1 zeigt das Schema eines Filtrationssystems, das unter dem Namen Amicon S1 im Handel ist.
• Fig. 2 zeigt eine Filtrationspatrone des Filtrationssystems von Fig. 1.

The invention is illustrated by the figures and the following examples.

• Fig. 1 shows the schematic of a filtration system, which is commercially available under the name Amicon S1.
• FIG. 2 shows a filtration cartridge of the filtration system of FIG. 1.

Figure 1 shows a filtration system for ultrafiltration of organic solutions. The solution is passed from a storage tank (not shown) via a line 3, which is provided with a circulation pump 5 and has a throttle valve 7, a pressure gauge 9, a shut-off valve 11 and a drain opening 13, via the inlet head 15 into the filtration arrangement 17. The filtration arrangement 17, which is provided with an inlet head 15 and an outlet head 19 and has clamps 21, contains a spiral cartridge 23. From the filtration arrangement 17, the filtrate is fed via a line 25, which has a shut-off valve 27, a drain opening 29, a pressure gauge 31 and has a back pressure valve 33, into a further reservoir (not shown).

FIG. 2 shows the filtration arrangement 117. The filtration arrangement 117 has an inlet head 115 which is provided with a pressure gauge 116 and a clamping device 121. A permeate port 126 leads into the inlet head. The filtration arrangement 117 contains a spiral cartridge 123. At the upper part of the filtration arrangement 117 there is the outlet head 119, which is provided with a back pressure valve 120 and a clamping device 121.

example 1

Test of a Sartorius polysulfone membrane with a molecular exclusion of 100,000 daltons for virus depletion

A filtration cassette made of polysulfone from Sartorius (Göttingen, Germany) was subjected to a phage suspension in tangential flow, the conditions described in the prototype process being observed. Table 1 shows the result of these test runs at three different partial pressures. The phage was only reduced by a power of ten at all partial pressures. In order to achieve a depletion by a factor of 10 ¹⁰ , the filtration would have to be repeated at least 10 times and this repetition had to be checked by exposure to the phage fr.

Example 2

For this example, virus depletion was tested by the Amicon S1 filtration system with an ultrafiltration membrane with a molecular exclusion of 30,000 daltons.

berechnet, wobei Pp der Permeatdruck bedeutet, der üblicherweise gleich Null und P_a ist. Die Proben wurden mit einer Schlauchquetschpumpe 31 bei 130 Umdrehungen pro Minute und einem Schlauchinnendurchmesser von 8 mm in die Patrone 23 gepumpt und der Transmissionsdruck durch Regulierung des Auslaßventils auf 0,2, 0,4 und 0,7 bar eingestellt.The technical principle of the filtration system is shown in Figure 1, the filtration cartridge is shown in Figure 2. The solution to be filtered flows in a tangential flow over the membrane and part of the solution is filtered off by the transmembrane pressure (P _t ) existing over the membrane. The pressure at inlet 15 (P _a ) and outlet 19 (P _b ) of the device can be read on two pressure gauges. The transmission pressure that forms over the membrane is calculated according to the formula

calculated, where Pp means the permeate pressure, which is usually zero and P _a . The samples were pumped into the cartridge 23 with a peristaltic pump 31 at 130 revolutions per minute and an inner tube diameter of 8 mm and the transmission pressure was adjusted to 0.2, 0.4 and 0.7 bar by regulating the outlet valve.

To test the cartridge, phage solutions with a titer of 7.8 x 10 ⁹ pfu (plaque forming units) per ml (in 10 mM Tris-CI buffer, pH 7.5 with 100 mM NaCl and 1 mg bovine serum albumin per ml) were added to the Pumped membrane. 1.5 l of the phage solution were filtered per test. Between each filtration, the cartridge was washed with 0.1 M NaOH and then rinsed with PBS (phosphate buffered saline) until the eluate was neutralized. This washing process completely inactivated phages remaining in the system. The cartridge was stored in 10 mM NaOH.

Table 2 contains the results of this test with the filtration cartridge SlY30 of the serial number 8864. There were discrepancies of 4.53 tens logarithms immediately after start-up and 4.4 tens logarithms after storage of the cartridge in 10 mM NaOH for several months after the first Filtration.

Example 3 Determination of the depletion rate in an ultrafiltration system with the bacteriophage fr

A spiral cartridge S1Y30 from Amicon with the serial no. 8864. For this purpose, 600 ml of a phage suspension with an initial titer of 3x10 ^{10 was} filtered three times in succession over the cartridge in five parallel batches. Between each filtration step, the cartridge was rinsed with 2 I RO water (water purified by reverse osmosis). Table 3 shows that in all five parallel runs after three filtrations through the cartridge, no infectious phage fr is contained in 1 ml of the permeate.

Example 4 Depletion of test viruses by a factor of 12

The SlY30 filtration cartridge with the serial number 10330 was tested. The phage solution consisted of 600 ml phage buffer (methods) with 600 mg bovine serum albumin and 50 ml phage concentrate (titer: 1.3x10 ¹² pfu (plaque forming units) / ml). The filtration was first carried out three times in succession. The volume of the diltrate decreases from 600 to 400 to 380 ml. The filtration times were approx. 20 minutes per step. Between each filtration, the cartridge was washed with 1 l of 10 mM sodium hydroxide solution to inactivate phage residues and then with dist. Rinsed water to neutrality (measured with the pH electrode).

The virus content in the individual filtrates was determined and is shown in Table 4.

Since no bacteriophage was detectable in 1 ml filtrate after the 2nd filtration, the last filtrate (380 ml) was again enriched with 40 ml virus concentrate (titer: 5.2x10 ¹² pfu / ml) and filtered three times in succession. As can be seen from Table 1, no phages can be detected in 1 ml of the filtrate after the second filtration.

The data from the virus depletion (Table 4) show that with the ultrafiltration cartridge used, the virus titer decreased by 6.92 and 7.22 tens logarithms after the first filtration. After the second filtration, no phages were detectable in the filtrate. Accordingly, the viruses were reduced by a total of 11 powers of ten after the first two filtrations and by 11.7 powers of ten after the further two filtrations, which arithmetically results in a total reduction of 22.7 powers of ten after four filtrations. The depletion of 12 to 16 that is often recommended in the literature is exceeded with 18.22 powers of ten after just three filtrations. The virus depletion is better in this production series with 7 logs of ten than in the production series used in Examples 2 and 3 with approx. 4.5 logs (tables 3 and 4). Significant differences in virus depletion can thus be determined between the individual production batches, which in turn underlines the need for careful validation with a test virus.

Example 5 Control of the virus depletion of a thymus extract by determination of BSA

The thymus was removed from calves and homogenized. An extract was obtained from this homogenate in a manner known per se. The bacteriophage fr (ATCC No. 15767-B1; Knolle and Hoffmann Verlag, Virology, Vol. 123, 271-273 (1964) was added to the extract thus obtained as a test virus, and the content of BSA and of purine and pyrimidine bases was determined using a HPLC analysis determined in a conventional manner known to those skilled in the art.

The sample with the test phage was then, as described in Examples 3 and 4, filtered through a filtration cartridge SlY30 with the serial number 10330 (Amicon Div .; WR Grace & Co .; Danvers, MA, USA) and both the virus depletion and the acceptance to BSA determines. The decrease in the virus correlated with the decrease in BSA.

Bacteriophage fr (ATCC 15767-B1) was deposited on November 19, 1964 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852-1776 USA, and has been freely available since November 19.

E. Coli 3300 (ATCC 19853) was deposited on January 12, 1967 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852-1776 USA, and has been freely available since January 12, 1967.

Claims (10)

1. Method to remove viruses and/or pathogenic material from organic preparations, characterised in that the material to be purified is passed through an ultrafilter or an ultrafiltration unit, the removal rate of which has previously been ascertained by admitting into the filter or filtration unit viruses of the leviviridae family or other comparatively small bacteriophages, by determining the titer of the viruses before and after the filtration, and by ascertaining the removal rate therefrom.

2. Method according to claim 1, characterised in that MS2, f2, f4, Qß, Vk, ST or R17 is used as the test virus.

3. Method according to one of claims 1 or 2, characterised in that the bacteriophage fr, ATCC No. 15767-B1, is used as the test virus.

4. Method according to one of the preceding claims, characterised in that the specimen containing the test virus is ultrafiltered, whilst adhering to precisely defined pressure conditions, and the same precisely defined conditions are adhered to for the purification of the organic material.

5. Method to determine the removal rate of viruses and/or pathogenic material in organic preparations, characterised in that a specimen of the material is mixed with viruses of the leviviridae group as the test virus, this specimen is subjected to the purification steps intended for the material, the number of viruses is determined before and after purification, and the decrease in the virus titer is detected.

6. Method according to one of the preceding claims, characterised in that the removal rate of a marker substance is determined, which substance was already contained in or subsequently added to the material to be purified, the ratio of the removal rates between the marker and the virus is formed, and the removal of the virus is tracked by the removal of the marker substance.

7. Method according to one of the preceding claims, characterised in that the material to be purified is obtained from plant parts, from human or animal tissues or organs, or from bacteria or virus extracts.

8. Method according to one of the preceding claims, characterised in that the material to be purified is obtained from spleen, thymus and/or bone marrow.

9. Method according to one of claims 6 to 8, characterised in that a protein or a nucleic acid is used as the marker substance.

10. Method according to one of claims 6 to 9, characterised in that BSA is used as the marker substance.

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